NAD+ (nicotinamide adenine dinucleotide) is an essential cofactor for many important NAD+-consuming enzymatic classes, such as sirtuins, poly ADP-ribose polymerases (PARPs), and cyclic ADP-ribose synthetases. As such, the bioavailable pools of NAD+ (the oxidized form of NAD) that regulate these critical enzymes represent links between metabolism, pathology, and numerous essential biological processes. The ability to monitor NAD+ levels in the cells is critical to understanding when, where, and how these enzymes function.
Sensors are available that can monitor NAD+/NADH ratios in a cell. However, NAD+ regulated enzymes operate in the nucleus and cytoplasm and are therefore unlikely to be regulated by redox reactions. Furthermore, NAD+ levels can be as much as 700-fold higher than NADH levels with concentrations in the micromolar range. Many NAD+ consuming enzymes have Km values in the micromolar range. Finally, current methods are unable to measure NAD+ concentrations in subcellular compartments and organelles. So directly monitoring NAD+ is key to understanding the function of NAD regulated enzymes.
Measurement of NAD+ using methods such as HPLC and mass spectrometry require harvesting and processing of cells and/or tissues. Using such methods, there is no way to differentiate the bioavailable pool of NAD+ from the protein-bound pool of NAD+ and certainly no way to measure intracellular localization of free NAD+ or changes in NAD+ levels over time.